Gentiopicroside promotes the osteogenesis of bone mesenchymal stem cells by modulation of β‐catenin‐BMP2 signalling pathway

Abstract Osteoporosis is characterized by increased bone fragility, and the drugs used at present to treat osteoporosis can cause adverse reactions. Gentiopicroside (GEN), a class of natural compounds with numerous biological activities such as anti‐resorptive properties and protective effects against bone loss. Therefore, the aim of this work was to explore the effect of GEN on bone mesenchymal stem cells (BMSCs) osteogenesis for a potential osteoporosis therapy. In vitro, BMSCs were exposed to GEN at different doses for 2 weeks, whereas in vivo, ovariectomized osteoporosis was established in mice and the therapeutic effect of GEN was evaluated for 3 months. Our results in vitro showed that GEN promoted the activity of alkaline phosphatase, increased the calcified nodules in BMSCs and up‐regulated the osteogenic factors (Runx2, OSX, OCN, OPN and BMP2). In vivo, GEN promoted the expression of Runx2, OCN and BMP2, increased the level of osteogenic parameters, and accelerated the osteogenesis of BMSCs by activating the BMP pathway and Wnt/β‐catenin pathway, effect that was inhibited using the BMP inhibitor Noggin and Wnt/β‐catenin inhibitor DKK1. Silencing the β‐catenin gene and BMP2 gene blocked the osteogenic differentiation induced by GEN in BMSCs. This block was also observed when only β‐catenin was silenced, although the knockout of BMP2 did not affect β‐catenin expression induced by GEN. Therefore, GEN promotes BMSC osteogenesis by regulating β‐catenin‐BMP signalling, providing a novel strategy in the treatment of osteoporosis.


| INTRODUC TI ON
Osteoporosis is a systemic disease characterized by bone loss, destruction of the bone microstructure and increased bone fragility, often leading to brittle fractures. 1 A large number of patients with osteoporosis not only suffer from severe pain, but they are also subjected to a heavy financial burden. 2 In particular, the bone resorption rate of postmenopausal women is significantly higher than that of osteogenesis, leading to a serious bone loss. 3 With the development of ageing population, the number of postmenopausal patients with osteoporosis is increasing year by year, causing a significant impact on the medical community and the whole society. 4 At present, the drugs used to treat postmenopausal osteoporosis play a role mainly by inhibiting bone absorption and promoting bone formation. 5,6 However, long-term use of anti-osteoporosis drugs can cause a series of adverse reactions, including myasthenia gravis, influenza-like diseases and gastrointestinal tumours. 7 Thus, it is of utmost importance to find a new alternative therapy to cure osteoporosis. The activity of osteoclasts and osteoblasts needs to be precisely coordinated to maintain skeletal integrity. 8 Osteoclasts are multinucleated giant cells derived from monocytes/macrophages, whose main function is to promote bone resorption. 9 Osteoblasts are mainly differentiated from bone mesenchymal stem cells (BMSCs) and deposited in the calcified bone matrix, which has a significant impact on the formation of a new bone. 10 The primary cause of osteoporosis is due to the decrease in osteoblasts that leads to a reduced bone formation, and the increase in osteoclasts resulting in an increased osteolysis. Recently, several reports pointed out that stimulating osteoblast differentiation may be an effective way to prevent and treat osteoporosis. [11][12][13] BMSCs are stem cells with a multi-directional differentiation, as they can differentiate into several cell types including chondrocytes, osteoblasts, adipocytes and endothelial cells under specific conditions. 14 BMSCs first differentiate into precursor osteoblasts, then into osteoblasts, and finally they gradually form the mature osteoblasts. 15 However, the osteogenic capability of BMSCs gradually decreases with the increase of old age, whereas the adipogenic capacity of BMSCs increased, leading to the down-regulation of bone formation and finally osteoporosis. 16 During the osteogenic differentiation, BMSCs release a series of osteogenic factors, including osteocalcin (OCN), osterix (OSX), osteopontin (OPN) and runt-related transcription factor 2 (Runx2), which accelerate the maturation of osteoblasts. 16,17 Therefore, BMSCs may be a suitable cell source for studying osteogenesis.
The Wnt pathway and BMP pathway occupy a critical position in the modulation of osteoblast differentiation. 18,19 When the frizzled transmembrane receptor binds to LRP5 and/or LRP6, it can induce the secretion of Wnts and activates the canonical Wnt pathway. 20 Subsequently, β-catenin is released and transcribed into the nucleus to regulate the generation of osteogenic markers. 21 The upregulation of BMP pathway induces the phosphorylation of Smad proteins. 22 Then, the activated Smad proteins actively regulate the transcription of osteogenic factors (Runx2 and OCN), thus promoting osteogenic differentiation. 23,24 GEN can be obtained from the natural plant Gentiana ManshuricaKitag, and it is a type of natural compounds with numerous biological activities such as anti-resorptive properties. It is widely used in the traditional Chinese medicine. GEN has a variety of pharmacological activities, including antioxidative, anti-inflammatory, antibacterial and anti-osteoporotic effects. [25][26][27] Recently, it was reported that GEN suppressed RANKL-induced osteoclastogenesis by regulating NF-κB and JNK signalling pathways and that it represented a potential drug in the treatment of osteoporosis. 28 Based on the above studies, our hypothesis is that GEN could induce osteogenic differentiation of BMSCs in vitro and bone formation in vivo.
Collectively, in order to understand the mechanism of action of GEN during bone formation, the effect of GEN on BMSCs function needs to be further characterized. Therefore, the purpose of this study was to find whether GEN could promote the osteogenic differentiation of BMSCs and explore the molecular mechanism induced by GEN in differentiating BMSCs. We used the ovariectomized (OVX) mouse model, the most popular animal model for postmenopausal osteoporosis, in which the acceleration of cancellous bone loss and the decrease of cortical bone are closely correlated to oestrogen deficiency. 17,29 Thus, this model is beneficial for us to explore the influence of GEN on bone density and osteogenic factor expression in vivo.

| MATERIAL S AND ME THODS
This project was carried out with the permission of the Ethics Committee of the Ningbo No. 6 Hospital (registered number 2015-018).

| Cell culture and treatments
Five female C57BL/6 mice (4-week-old) were used and killed to obtain BMSCs cultured according to a previous work. 30 In brief, the mouse femur was collected and the surrounding soft tissues were removed. Then, the bone marrow in the femur was washed three times with α-MEM (Sigma-Aldrich). The obtained bone marrow content was placed in a dish containing a complete medium(Sigma-Aldrich). BMSCs were incubated at 37°C in a 5% carbon dioxide incubator. The third generation of BMSCs was used for our experiments. When the confluence reached 70%, the osteogenic medium was added to allow the osteogenic differentiation. The osteogenic medium consisted of complete medium supplemented with 0.1 mM dexamethasone (Sigma-Aldrich), 5 mM β-glycerophosphate (Sigma-Aldrich) and 100 mM ascorbic acid (Sigma-Aldrich). Then, BMSCs were cultured for 14 days under the osteogenic environment, changing the medium every 3 days.
The silencing of β-catenin and BMP 2 gene was performed according to previous investigations. 31,32 The transfection sequences were the following: β-catenin, former primer Next, the cells cultured in the osteogenic medium and transferred with the Ad-Cre (at a concentration of 5 × 10 8 pfu/mL) for 48 hours.
Ad-GFP was used as a control. Next, the cells were treated with 40 μM GEN in the osteogenic medium for 7 days. Finally, the expression of Runx2, OSX, OCN, OPN, BMP2 and β-catenin was evaluated by q-PCR and Western blotting. Next, BMSCs were fixed with formalin for 10 minutes, ALP staining buffer (Sigma-Aldrich) was added, and the cells were incubated for 30 minutes at room temperature.

| Alizarin red staining
BMSCs (2.08 × 10 4 cells/cm 2 ) were seeded into 6-well plates and treated with GEN at different concentrations (0, 10, 20 and 40 μM) for 14 days under osteogenic environment. Next, the medium was discarded, the cells were washed with PBS for three times, and a formalin solution was added to fix the cells. After 20 minutes, the formalin was discarded, and BMSCs were washed 2 times with medium and treated with alizarin red solution (Sigma-Aldrich) for 30 minutes.
Subsequently, the stained cells were observed under an optic microscope and images were taken in random fields. Finally, the software Image J (NIH, Bethesda, MA, USA) was used to perform the statistics of the mineralized nodules, and the nodules larger than 0.04 mm were included in the statistical calculation. 33

| Western blot
BMSCs (2.08 × 10 4 cells/cm 2 ) were seeded into 6-well plates and treated with GEN at different concentrations (0, 10, 20 and 40 μM) in the osteogenic medium for 14 days. To assess the influence of GEN on the signalling of BMP and Wnt/β-catenin, BMSCs were treated with either 300 ng/mL Noggin (Sigma-Aldrich) 34 or 100 ng/ ml DKK-1 (Sigma-Aldrich) 35 in the treatment with GEN for 2 weeks.
After the treatment, the total BMSC proteins were extracted by ra- The target proteins were first corrected over β-actin expression and then as fold change from control group.

| Experimental model and animal groups
Thirty-six female C57BL/6 mice (8-week-old, 21 ± 2 g) were ob- The OVX + GEN groups were treated by an oral gavage of 50 mg/ kg/day GEN during these 3 months. The Sham group and OVX group received the same dose of saline by oral gavage. After 3 months, the experimental mice were killed by cervical dislocation and the femurs were collected for further studies.

| Histological and immunohistochemical staining
The femur was immersed in 4% paraformaldehyde for 48 hours and decalcified using 15% ethylenediaminetetraacetic acid for 14 days. Then, they were dehydrated, paraffin embedded and cut

| Microcomputer tomography analysis
The collected femurs were preserved in 4% paraformaldehyde for 48 hours. The prepared femur was scanned and analysed by high-resolution micro-CT (Caskaisheng, China). The scanning parameters of the micro-CT were set as follows: 80 kV, 15 μA and a scanning thickness of 20 μm. The area below the crud end of femoral shaft was chosen as the analysis area for statistical analysis. 36 The bone parameters for statistical analysis included the following three indexes: trabecular bone mineral density (BMD), trabecular number and trabecular thickness.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 6 (Manufacturer, La Jolla, CA, USA). All in vitro experiments were repeated three times, and each experiment was carried out in triplicate. In the in vivo experiments, each group contained at least 6 rats.
Results were expressed as mean ± standard deviation (SD). One-way ANOVA and Dunnett's test were used to compare multiple groups, whereas unpaired Student's t test was used for the comparison of two groups. P <.05 was considered statistically significant.

| Effect of GEN on BMSC proliferation
The chemical structure of GEN is shown in Figure 1A. The proliferation of BMSCs treated with GEN (10-40 μM) was not significantly changed ( Figure 1B). However, 80 μM GEN significantly inhibited the proliferation of BMSCs (less than 1-fold, P <.05). Thus, GEN was not harmful to BMSCs at the concentrations of 10, 20 and 40 μM.

| GEN strengthens the osteogenic differentiation in BMSCs
The effect of GEN alone on BMSCs was tested as first. The osteogenic differentiation of the BMSCs treated with GEN for 14 days without osteogenic medium was not significantly affected ( Figure S1

| GEN promotes bone formation in OVX mice
The OVX mouse model was used to confirm these in vitro results.
The OVX osteoporosis mouse model is the most used animal model in studying postmenopausal osteoporosis. Ovariectomy can cause bone loss acceleration and cortical bone formation reduction, which are closely related to oestrogen deficiency. 29,37 To test the effect of the ovariectomy, the body weight and the mass of the uterus were measured ( Figure S2)(P <.05). The body weight of the OVX group and OVX + GEN group was greater than that of the Sham group  Figure S2A) (less than 1-fold, P <.05). In contrast, the mass of the uterus in the OVX group and OVX + GEN group was less than that in the Sham group ( Figure S2B) (less than 3-fold, P <.05).
The histopathological images of all groups (sham group, OVX group, and OVX + GEN group) are shown in Figure 4. HE staining results showed that the number of bone trabeculae in the OVX group was significantly less than that in the Sham group, whereas the number of bone trabeculae in the OVX + GEN group was higher than that in the OVX group, but no significant difference was observed between the Sham group and OVX + GEN group ( Figure 4A). The results of micro-CT showed that the BMD, the number of trabeculae and the thickness of the trabeculae in OVX + GEN group were higher than those in the OVX group, whereas the same parameters in the OVX group were remarkably lower than those in the Sham group ( Figure 4B-E) (P <.05). However, no statistical difference was found between OVX + GEN group and Sham group. Runx2 expression in the OVX group was less than that in the Sham group, whereas its expression in the OVX + GEN group was higher than that in the OVX group ( Figure 5A) (nearly 2-fold, P <.05). Similar to the results of Runx2, the expression of OCN ( Figure 5B) (less than 3-fold, P <.05) and BMP2 ( Figure 5C) (nearly 3-fold, P <.05) in OVX group was lower than that in the Sham group and OVX + GEN group. Thus, our results demonstrate that GEN effectively promoted osteogenesis in OVX osteoporotic mice and showed a good antiosteoporotic effect.

| BMP pathway and Wnt/β-catenin pathway activated by GEN in BMSCs
As the BMP signalling 38

| GEN-induced osteogenic differentiation is a β-catenin-BMP2-dependent effect
To further reveal the specific mechanism of GEN in regulating of BMP signalling and Wnt/β-catenin signalling, gene silencing was performed in vitro. The transfection with Ad-Cre efficiently silenced the β-catenin ( Figure 8A) and BMP2 ( Figure 8G)  In this study, GEN was found to be able to activate osteogenic differentiation in BMSCs under osteogenic medium. However, there was no effective influence on BMSCs without osteogenic condition.
These results suggest that GEN has no significant effect on nor- Taken together the results in the current study, a model illustrating the potential mechanism used by GEN to promote the osteogenic differentiation of BMSCs could be proposed. GEN stimulates osteogenesis by increasing ALP, Runx2, OSX, OCN and OPN through the activation of the Wnt/β-catenin-BMP2 signalling, thereby promoting the differentiation of BMSCs into osteoblasts ( Figure 9).
This study has some limitations. The effect of GEN on human BMSCs from healthy persons and osteoporosis patients would be further to identify our findings.

| CON CLUS IONS
In conclusion, this study provided novel insights into the effect of GEN on BMSCs osteogenic differentiation and its protective effect against bone loss. Although further studies are required to confirm these results, GEN might represent a promising approach in the treatment of osteoporosis.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no conflict of interest.

F I G U R E 9
Proposed model depicting the underlying mechanisms of GEN in promoting the osteogenic differentiation of BMSCs